Resistant and/or relapsed acute myeloid leukemia (AML) remains a significant unmet clinical need for which the field needs new and more effective therapeutic strategies based on further advances in modeling leukemogenesis. Loss of function (LOF) RUNX1 mutations, which are associated with worsened prognosis, occur frequently in AML and myelodysplastic syndrome (MDS). Murine Runx1-/- hematopoietic stem and progenitor cells (HSPCs) have features such as decreased proliferation, apoptosis, and biosynthetic capacity, which promote long-term persistence. RUNX1 mutations alone are insufficient to cause MDS/AML and must occur in the context of additional mutations, which may partially be explained by the slow growth phenotype observed in Runx1-/- HSPCs. However, the mechanisms by which cooperating mutations alter the properties of RUNX1 mutant HSPCs are currently unknown. Most of the mutations that co-occur with RUNX1 in MDS/AML involve genes encoding epigenetic modifiers of chromatin and DNA, such as ASXL1, EZH2, TET2, IDH1/2, & DNMT3A. This finding suggests that epigenetic alterations are a common mechanism by which leukemogenesis is supported in the context of a RUNX1 mutation. We aim to determine how cooperating mutations change the phenotypes of Runx1-/- HSPCs and the mechanisms by which this occurs. In Aim1, we will characterize the phenotypic consequences of concurrent mutations in Runx1 and Asxl1. ASXL1 is the most frequently co-mutated gene with RUNX1 in MDS/AML. Notably, the phenotypes of murine Asxl1-/- HSPCs (e.g. increased proliferation and apoptosis) are opposite of Runx1-/- HSPC phenotypes. We will generate Runx1-/-Asxl1-/- double knockout mice and examine the proliferative, apoptotic, and biosynthetic characteristics of HSPCs. Functionally, both Asxl1 and Runx1 act to recruit repressive polycomb group (PcG) complexes to genomic loci. We hypothesize that altered recruitment of PcG complexes results in dysregulated transcriptional activation at multiple loci encoding genes involved in MDS/AML progression. To test this hypothesis, we will generate and correlate RNA-seq data with ChIP-seq data for chromatin marks in Runx1-/- Asxl1-/- HSPCs to identify differentially regulated pathways. In Aim 2, we will use traditional genetic mouse models as well as a CRISPR-based screening platform to assess the leukemogenic potential of combinations of Asxl1, Ezh2, Tet2, and Dnmt3a mutations in the context of LOF Runx1 mutations. We will determine which combinations are potent contributors to MDS/AML progression and the mechanisms by which they cooperate. This work will provide new insights into modeling leukemogenesis and potentially identify novel therapeutic targets.